Method for identifying and for extracting endotoxin

ABSTRACT

The invention relates to a method for identifying endotoxins for eliminating said endotoxins from a sample, with the aid of bacteriophage tail proteins.

The present invention relates to a method for detecting and fordepleting endotoxins from a sample.

Endotoxin (ET) describes a family of lipopolysaccharides which togetherwith proteins and phospholipids form the outer cell wall ofGram-negative bacteria. Endotoxins occur exclusively in this bacterialgroup and play an important role in the organisation, stability andbarrier function of the outer membrane. Numerous bacteriophages useendotoxin or general lipopolysaccharide for specific detection of theirhost bacteria.

All endotoxin variants comprise a heteropolysaccharide which is bondedcovalently to lipid A (Holst, O., 1999, Chemical structure of the coreregion of lipopolysaccharides. In: Endotoxin in health and disease(Brade, H., Morrison, D. C., Opal, S., Vogel, S. eds.), Marcel DekkerInc. New York). Lipid A anchors endotoxin in the outer bacterialmembrane. The heteropolysaccharide, which comprises a coreoligosaccharide and the O antigen, appears in the surrounding solutionand determines the serological identity of the bacterium. The O antigencomprises repetitive oligosaccharide units, the composition of which isstrain-specific (see in this context Holst et al., above).Characteristic building blocks of the core oligosaccharide are2-keto-3-deoxyoctonate (KDO) and L-glycero-D-mannoheptose (Hep).

The most conservative part of endotoxin of different types is the lipidA. The inner core region is preserved similarly to lipid A, the outercore region already has a higher variation. The inner core region, KDOand lipid A itself carry a plurality of phosphate groups as substituentsand are therefore responsible for the negative charge of endotoxin.Furthermore, the phosphate groups on the lipid A and on the core regioncan be substituted variably with arabinose, ethanolamine and phosphate.Individual saccharide building blocks of the O antigen are acetylated,sialated or glycosylated. The O antigen varies in addition with respectto the number of repetitive units, for which reason the endotoxinpopulation of each bacterium has a certain heterogeneity (Palva E. T.,Makela P. H., Lipopolysaccharide heterogeneity in Salmonella typhimuriumanalysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.Eur J Biochem. 1980;107(1):137-43; Goldman R. C., Leive L.,Heterogeneity of antigenic-side-chain length in lipopolysaccharide fromEscherichia coli 0111 and Salmonella typhimurium LT2., Eur J Biochem.1980;107(1):145-53).

Endotoxins are biomolecules which can be found in practically allaqueous solutions without corresponding precautionary measures.Endotoxins in humans and animals can lead to sepsis, to a strongincorrect response of the immune system. Hence, for example whenproducing pharmaproteins, contamination with endotoxin should bedetected precisely and should be removed completely subsequently.Endotoxin represents a problem with genetically engineeredpharmaceuticals, gene therapeutics or substances, which are injectedinto humans or animals (e.g. veterinary treatment or in animal tests).However, not only in medicinal but also in research applications, suchas transfection experiments of mammal cells, inhibition or lowering ofthe transfection efficiency by means of endotoxin can be observed.

In order to be able to use proteins within the framework of clinicalstudies, the European and American pharmacopoeia demand that theproteins fall below specific boundary values for endotoxin level (e.g.immune serum globulin 0.91 EU/ml, this corresponds to 5 EU/kg bodyweightand hour (dosage=EU/kg*h); EU=endotoxin unit; FDA (Food and DrugAdministration): Guideline on Validation of LAL as End Product). If amedicine or proteins contained therein have too high an endotoxin level,this can lead to the death of the experimentee. The misdirected immunedefence damages the patient due to overreaction. This can lead to tissueinflammation, drop in blood pressure, heart racing, thrombosis, shocketc. Even a longer enduring endotoxin exposition in picogram quantitiescan lead to chronic side effects, such as e.g. immune deficiences,septic symptoms etc. Within the framework of substance production, inparticular in processes with “good manufacturing practice” (GMP)conditions, it is therefore attempted to deplete endotoxin as far aspossible. However, endotoxin removal in proteins, polysaccharides andDNA is problematic. In the case of proteins themselves, there are largeproblems due to their intrinsic properties, such as charge state orhydrophobicity, which can virtually prevent endotoxin removal or canlead to large product losses in the removal procedure.

At present, only three methods for endotoxin detection in biologicalsolutions are described, only the first two methods being permitted bythe FDA. 1. “Rabbit Pyrogen Testing”; a method in which a living rabbitis injected with an endotoxin solution and hence an immune reaction istriggered. This endotoxin-induced immune response is detected by thedevelopment of fever. 2. The “Limulus Amoebocyte Lysate (LAL)”—Test, thetest which is used most frequently at present (Bio Whittacker, Inc.,Charles-River, Inc., Associates of Cape Cod, Inc., all USA), can bestandardised in a significantly improved way. With this method, theagglomeration of the blood of the horseshoe crab (Limulus polyphemus) ismeasured after endotoxin contact. 3. A further possibility is the use ofa special cell culture system (Sterogene Inc., USA) with whichactivation of monocytes is tracked via the appearance of specificcytokines.

The two first-mentioned methods are however very expensive (cf.Competitive comparison endotoxin detection) and, due to the largerequirement for test animals or for blood of the very rare horseshoecrab, are dubious not least on the grounds of animal protection. The LALtest can in fact also be miniaturised and automated but, due to lowstability of the components, has huge disadvantages in application. Oncea LAL solution has been opened it must be processed and used upimmediately since the components aggregate within a few hours. Skilledpersonnel are required for all test methods and the methods are verysusceptible to interference, because for example the immune system ofrabbits can react entirely differently to the same dose of endotoxin.The cell culture method of the Sterogene Company, like all cell culturemethods, is likewise very complex and has problems with respect tostandardisation.

It can be established overall that there is no easily handled economicalmethod for endotoxin detection and the methods used at present have aseries of disadvantages. There is therefore a requirement for a methodwhich avoids these disadvantages.

There is in general a series of methods for endotoxin depletion frombiological solutions. Particularly in the case of proteins, there havehowever to date been no generally applicable standard methods. Therespectively used methods are adapted to the specific properties of therespective protein and to the corresponding production process of theprotein. There are various possibilities for endotoxin depletion, eachof these methods having specific advantages and disadvantages.

Ultrafiltration (Petsch, D. & Anspach, F. B., 2000, J. Biotechnol. 76,97-119 and references therein) is used for endotoxin depletions fromwater and solutions with low-molecular components, such as salts, sugarsand antibiotics but is not suitable for high-molecular proteins or DNA.

2-phase extraction (e.g. WO 0166718, Merck) is intended to separatewater-soluble proteins and DNA from endotoxin but produces detergentresidues in the purified product. The method is in additiontime-consuming due to multiple repetition of the purification procedure.

An anion exchanger (DEAE) method is used likewise for endotoxindepletion from DNA and basic proteins (e.g. U.S. Pat. No. 5,990,301,Qiagen; WO 9414837, Enzon) but requires a low ionic strength (<50 mMNaCl) and leads to a protein co-adsorption in the case of acidicproteins.

A further method for endotoxin depletion from DNA and proteins (e.g.BSA, myoglobin, gamma-globulin, cytochrome C) is affinity-adsorption(e.g. polymyxin B, histamine, histidine, polylysine) e.g. GB 2192633(Hammersmith Hospital) which is however toxic in the case of polymyxin Band can lead to co-adsorption of proteins in the case of low ionicstrengths.

Furthermore, immune-affinity-chromatography is used, the specificity forspecific endotoxins being able to be achieved only via expensiveantibodies (U.S. Pat. No. 5,179,018, Centocor; WO 0008463, Bioserv)against core oligosaccharide.

Furthermore, the S3delta-peptide (WO 0127289) of the factor C (acomponent of the LAL test) (WO 9915676, both: National University ofSingapore) is used with proteins (e.g. BSA, chymotrypsinogen), thismethod having however low efficiency in the case of high ionic strengthsand high production costs are also involved (production in insect cellculture).

In application in the pharmaceutical industry, essentially three methodsare found for protein solutions adapted to the properties of the targetproteins;

-   -   anion exchanger chromatography    -   reversed-phase chromatography; this has the disadvantage that it        is not equally suitable for all proteins—in particular is        problematic in the case of hydrophobic proteins. This method is        furthermore very time-consuming.    -   Rem Tox (Millipore Company): this method has the disadvantage        that, in addition to a very long incubation duration, the        non-specific binding component is high and the protein retrieval        is often not adequate.

A rough endotoxin depletion of proteins to a value up to 10 EU/ml ispossible in many cases with the existing methods. The remainingconcentration of endotoxin however always still has a toxic effect. Afurther depletion (=fine purification) is therefore offered or,dependent upon the dose of the protein in the medical application, isprescribed as mandatory by the European pharmacopoeia (e.g 5 EU/kgbodyweight and hour in intravenous applications) and by the FDA.However, this fine purification is often not ensured satisfactorily withpresent methods. The current market methods have here significantdisadvantages and, in the case of specific proteins, often cannot beapplied or only with considerable losses of the target protein.

The object underlying the invention is therefore to provide a methodwhich can detect endotoxins in samples. Furthermore, the objectunderlying the invention is to provide a method with which endotoxinscan be removed from aqueous solutions.

The objects are achieved by the subject defined in the patent claims.

The subsequent Figures explain the invention.

FIG. 1 shows a schematic overview of the chemical structure of endotoxinfrom E. coli O111 :B4. Hep=L-glycero-D-mannoheptose; Gal=galactose;Glc=glucose; KDO=2-keto-3-deoxyoctonate; NGa=N-acetyl-galactosamine;NGc=N-acetylglucosamine.

FIG. 2 shows the results of tests with chromatography columns whichcarry NStrepS3Cp12 immobilised via sulfhydryl radicals. (A) Endotoxinremoval from protein solutions: bovine serum albumin (BSA), carbonicanhydrase (CA) and lysozyme (Lys) were incubated for 1 h on the columnand subsequently eluted with buffer. The endotoxin concentration beforeand after the column was measured with the LAL test and the percentageremoval was calculated therefrom. (B) Protein retrieval: the proteinconcentrations of the starter solutions and the fractions after thecolumn were determined by absorption measurement at 280 nm and thepercentage protein retrieval was determined therefrom.

FIG. 3 shows the endotoxin removal from a lysozyme solution viachromatography columns with “undirected” (1) and “directed” (2)immobilised p12. In both cases, p12S3C was bonded to NHS-activatedcolumns. The “undirected” immobilisation was effected via primary aminoradicals of p12S3C, which produce covalent compounds with the carriersubstance by reaction with the NHS groups. A “directed” cross-linking ofp12S3C via an N-terminal cysteine is achieved by diamino ethane and SIA(N-succinimidyl-iodoacetate). (A) Percentage endotoxin removal. (B)Protein retrieval.

FIG. 4 shows the results of tests with biotinylated p12 which was bondedto magnetic beads via streptavidin. (A) Endotoxin depletion from buffer(20 mM hepes, 150 mM NaCl, pH 7.5) and protein solutions was determinedby means of LAL test. (B) The protein retrieval was determined for theprotein solutions by absorption measurements. The separation of thebeads from the solution was effected by means of a magnet separator.BSA: bovine serum albumin. CA: carbonic anhydrase. Lys: lysozyme.

FIG. 5 shows the results of the endotoxin removal with p12 which wasimmobilised on agarose beads via biotin-streptavidin interactions. Theseparation of the immobilised p12 was effected by centrifugation. Theendotoxin removal from buffer (20 mM tris, 150 mM NaCl, pH 8.0) and BSAsolutions was determined by means of the endotoxin concentrations ofstarter solution and residue.

FIG. 6 shows results of surface-plasmon-resonance measurements. (A)Resonance curves which were measured as response to injection of various(respectively in μg/ml: 100; 25; 6.25; 4; 1.56; 0.4) p12 concentrations(______). Binding is effected on endotoxin from E. coli D21fl which wasimmobilised on a hydrophobic HPA chip. The injection of p12 and EDTA (5mM) is marked via bars over the curves. Buffer: 20 mM tris, 150 mM NaCl,pH 8.0. (B) Equilibrium resonance values for the binding of p12 toimmobilised endotoxin were measured approximately 600 s after thebeginning of the p12 injection and plotted against the associated p12concentration. The continuous line shows a fit of the Langmuiradsorption isotherms (RU=RU_(max)*[p12]/[p12]+K_(d))) to the data. (C)Binding of E. coli to biotinylated p12 which was immobilised onstreptavidin chips. E. coli D21 e8 (______), the inner core region ofwhich is complete, to p12. In contrast, E. coli D21f2 (—), which has agreatly shortened core region, does not bind to p12. The measurementswere implemented in PBS.

FIG. 7 shows schematically the structure of the endotoxin core region ofvarious E. coli mutants.

FIG. 8 shows schematically the result of an endotoxin depletion by meansof chromatography column throughflow methods. E means equilibrationbuffer (20 mM hepes, 150 mM NaCl, 0.1 mM CaCl₂, pH 7.5), A means washingbuffer A (20 mM hepes, 150 mM NaCl, 0.1 mM CaCl₂, pH 7.5), B meanselution buffer B (20 mM hepes, 150 mM NaCl, 2 mM EDTA, pH 7.5), C meansregeneration buffer C (20 mM hepes, 150 mM NaCl, 2 mM EDTA, 0.005%NaDOC, pH 7.5), S means concentration of protein and endotoxin in thestarter solution. BSA means bovine serum albumin. EU means endotoxinunits. After injection (I) of 4 ml of the starter solution (S),re-rinsing took place with 15 ml washing buffer and the throughflow wasfractionated (respectively 2.5 ml during application, respectively 2 mlduring washing). Subsequently, the column was regenerated with thebuffers B and C and the discharge was collected likewise in fractions(respectively 2 ml). As is evident in the Figure, the BSA could be foundin the first 3-5 fractions after the injection. The content of endotoxinin these fractions was lower by the factor 100 than in the startersolution. The endotoxin bonded to the column was then washed from thecolumn with the buffers B and C.

FIG. 9 shows schematically the results of the endotoxin removal fromslightly contaminated buffer solution (5 EU/ml) in the throughflowmethod. p12 was immobilised (8mg p12/1 ml sepharose), undirected towardsNHS-activated sepharose 4 FastFlow (Amersham Biosciences, Uppsala,Sweden) and 3 columns were filled with respectively 2 ml column volumes.The experiment was implemented in parallel on 3 columns. Prior to theapplication of the sample, respectively 1 ml equilibration buffer (20 mMhepes, 150 mM NaCl, 0.1 mM CaCl₂, pH 7.5) was collected, thereafter thesample (S: endotoxin from E. coli O55:B5 in equilibration buffer, 4.6EU/ml) was injected (I) and the fractions of 5 ml and 2 ml werecollected. The regeneration of the column was effected by the additionof 4 ml regeneration buffer (B: 20 mM hepes, 150 mM NaCl, 2 mM EDTA,0.005% NaDOC, pH 7.5). The endotoxin concentration was determined bymeans of the LAL test (kinetically chromogenic LAL test, Charles-RiverInc.). The endotoxin impurities were able to be removed completely inall three experiments, i.e. the endotoxin concentration in thethroughflow was below the detection limit (<0.005 EU/ml).

The term “endotoxin depletion” as used here means complete or partialremoval of endotoxin from sample material.

The term “endotoxin” as used here describes bacterial lipopolysaccharidewhich is a component of the outer membrane of Gram-negative bacteria.

The term “bacteriophage tail protein” as used here describes thoseproteins which occur in bacteriophages and can bind components of cellmembranes. Normally, these proteins are localised in the bacteriophagetail but can also be localised on the bacteriophage head or on thenormal bacterial shell in the case of bacteriophages without a tail. Thecell components bonded by the bacteriophage tail protein detect inparticular endotoxins.

The term “non-specific immobilisation” or “undirected immobilisation” asused here means that coupling of a protein to a matrix is effected viaprotein radicals (primary amines) which are distributed over the entireprotein surface. The choice of group used for the coupling of theindividual protein molecule is random.

The term “directed immobilisation” as used here means that coupling iseffected via amino acid radicals or other radicals (e.g. glycosylationsof the protein), the position of which in the protein (e.g. N- orC-terminal) is known. The choice of these groups for the coupling iseffected by the choice of suitable reaction partners/linkers which reactpreferably with these radicals (e.g. coupling of sulfhydryl radicals toiodoacetate radicals; iodoacetate reacts a thousand times more quicklywith sulfhydryl radicals than with amino radicals).

The present invention relates to a method for detecting endotoxin,comprising the steps:

-   a) incubation of a sample with a bacteriophage tail protein,-   b) detection of endotoxin bonded to bacteriophage tail proteins.

The invention relates preferably to a method, in which the detection isimplemented by means of spectroscopic methods, e.g. fluorescenceemission, fluorescence polarisation, absorption or circular dichroism,or by means of capacitance measurement, e.g. electrical signals orindirectly by means of competition detection.

If necessary, after step a) and before step b), an additional step a′),separation of bacteriophage tail protein-endotoxin complex from thesample, is introduced.

The present invention relates furthermore to a method for removingendotoxin from a sample, comprising the steps:

-   a) incubation of a sample with or bringing a sample into contact    with bacteriophage tail proteins which are immobilised on a fixed    carrier, in a non-specific or directed manner,-   b) separation of the bacteriophage tail protein-endotoxin complex    from the sample.

Preferably, the ion composition of the bivalent ions, e.g. Ca²⁺, Mg²⁺and/or the pH value is adjusted before incubation in order to obtain anoptimal endotoxin-bacteriophage tail protein binding. Furthermore,during or after incubation, “demasking” of the bonded endotoxin byaddition of detergents and/or salts, e.g. Tween, triton NaCl or ammoniumsulphate or other substances, e.g. chitosan, sugar or lipids, whichaccelerate detachment of the endotoxins from e.g. proteins or nucleicacids, is preferred.

The bacteriophage tail protein can be naturally occurring or bemolecular-biologically or biochemically modified. The bacteriophage tailprotein can be modified by genetic engineering and/or biochemically forvarious reasons. For the methods according to the invention, not onlythe naturally occurring bacteriophage tail proteins can however be used,but also their variants. In the sense of the present invention, variantsmeans that the bacteriophage tail proteins have an altered amino acidsequence. These can be obtained by screening of the naturally occurringvariants or by random mutagenesis or targeted mutagenesis, but also bychemical modification. The bacteriophage tail proteins used for themethods according to the invention can be adapted by targeted or randommutagenesis in their specificity or their binding properties to carrierstructures. This binding to the carriers can be effected permanently,e.g. covalently or via a specific or non-specific biotinylation, butalso can be effected reversibly, e.g. via a reducible disulfide bridge.Furthermore, the stability can be increased by a modification. By meansof the molecular-biological or chemical mutagenesis, mutations areintroduced which can be amino acid additions, -deletions, -substitutionsor chemical modifications. These mutations can effect a change in theamino acid sequence in the binding region of the bacteriophage tailproteins, with the aim of adapting specificity and binding affinity totest requirements, e.g. increasing the binding of the endotoxins to thebacteriophage tail proteins or making them irreversible in order toimprove detection or depletion. Furthermore, a genetically engineered orbiochemical modification of the phage proteins can be implemented withthe aim of switching off the possibly present enzymatic activity inorder consequently to improve the binding or to make it irreversible.Furthermore, a genetically engineered or chemical modification of thephage proteins can be implemented in order to adapt the present physicalproperties of the protein, such as solubility, thermal stability etc.,in the sense of the method according to the invention.

Work to explain the three-dimensional structure of T4 p12 had shownthat, at increased temperature, proteolytic fragments of 33 kDa and 45kDa can be produced, the N- and C-terminal (33 kDa) or only N-terminal(45 kDa) are shortened. In contrast to the 33 kDa fragment, the 45 kDafragment is still able to bind to bacteria. Consequently, the C-terminusis involved in the cell binding.

The modification can furthermore have the purpose in particular ofenabling direct detection, e.g. by means of measurement of thetryptophan fluorescence. For example p12 has five tryptophan radicals.The fluorescence spectrum of the native protein indicates that theseradicals are extensively solvent-inaccessible. It is known from amultiplicity of scientific works that aromatic amino acids are almostalways involved in the binding of sugar radicals, as occur also inendotoxin. The binding of the sugar radicals to proteins can be followedby a quench of the tryptophan fluorescence or if necessary also inaddition by changing the fluorescence maximum. It can be supposed fromsome works that the unfavourable distribution of the fluorophores ofnatural p12 prevents exploitation of the fluorescent properties of p12for binding measurement. The fluorescence properties of p12 aredominated by the five tryptophan radicals, the fluorescence of which isaltered by the addition of endotoxin in a non-measurable manner. It isexpected from these data that rather tyrosine radicals are involved astryptophan radicals in the binding, the signal alteration of whichcannot be made visible in front of the high tryptophan background. Onthe basis of the proteolysis results, six tyrosines on the C-terminus ofp12 are possible for the endotoxin detection kit which can be madecorrespondingly “visible”. By means of a selective molecular-biologicalexchange of the five tryptophan radicals for tyrosines, thespectroscopic properties are specifically altered in a first step suchthat the endotoxin binding by fluorescence signal alteration of a singletryptophan radical is measurable. Subsequently, by means of a specificexchange of respectively one of the six tyrosines in the C-terminalregion for a tryptophan radical, the intensity of the measurable signalis significantly increased in order to obtain attractive signaldifferences for the development of an endotoxin-detection kit.

The bacteriophage tail proteins which are used depends upon whichendotoxins are intended to be detected or drawn off. Even now, a largenumber of known bacteriophages is available for a large part of thepreviously described bacteria and can be used for the methods accordingto the invention. The phages and the corresponding host bacteria areinter alia obtainable in the case of the following strain collections:ATCC (USA), DSMZ (Germany), UKNCC (Great Britain), NCCB (Netherlands)and MAFF (Japan).

Preferably, the bacteriophage tail proteins for the methods according tothe invention stem from bacteriophages, the host bacteria of which haverelevant significance with respect to medicine or biotechnology, such ase.g. E. coli which is used in the production of recombinant proteins orof nucleic acids for gene therapy. The bacteriophage tail proteins whichbind highly conserved regions of endotoxin, such as e.g. the core regionor lipid A, are particularly preferred. In particular, p12 andp12-similar bacteriophage tail proteins are preferred. In a combinationof endotoxin impurities from various host bacteria, a combination of thecorresponding endotoxin-detecting bacteriophage tail proteins can beused.

The detection or the depletion of endotoxin in or from a sample iseffected via the binding of endotoxin to the bacteriophage tailproteins. This binding can be detected for example by direct measurementby means of spectroscopic methods, e.g via fluorescence emission,fluorescence polarisation, absorption or circular dichroism.Furthermore, the binding can be made visible by electrical signals, e.g.a capacitance measurement. Furthermore, the binding of endotoxin to thebacteriophage tail proteins can also be detected indirectly viadisplacement experiments.

For the detection according to the invention, the bacteriophage tailproteins, if separation of the bacteriophage tail protein-endotoxincomplexes from the sample is required, can be coupled to suitablecarrier structures, e.g. magnetic particles, agarose particles,microtitre plates, filter materials or throughflow cell chambers(indirect detection). The carrier structures can comprise for examplepolystyrene, polypropylene, polycarbonate, PMMA, cellulose acetate,nitrocellulose, glass, silicon or agarose. The coupling can be achievedfor example by adsorption or covalent binding.

For the depletion method according to the invention, the bacteriophagetail proteins are coupled to permanent carriers. The permanent carrierscan be materials for chromatography columns (e.g. sepharose materials),filtration media, glass particles, magnetic particles, centrifugation-or sedimentation materials (e.g. agarose particles).

Functional coupling is hereby important, i.e. bacteriophage tailproteins, despite binding to the carrier material, have structures whichare accessible for endotoxin. The coupling of the bacteriophage tailproteins can be effected non-specifically or else preferably directed,via for example a selective biotinylation or coupled or via a spacer orlinker.

For this purpose, the bacteriophage tail proteins can be cross-linkedwith low-molecular substances, e.g. biotin, in order to bind via theselow-molecular substances to polypeptides, e.g. streptavidin, which fortheir part were immobilised on the carrier. Instead of biotin, theso-called Strep-tag (Skerra, A. & Schmidt, T. G. M. BiomolecularEngineering 16 (1999), 79-86) can furthermore be used, which is a shortamino acid sequence and binds to streptavidin. Furthermore, the His-tagcan be used which, via bivalent ions (zinc or nickel) or an antibodyspecific for it (Qiagen GmbH, Hilden), can bind to a carrier material.The Strep-tag and the His-tag are bonded preferably via DNArecombination technology to the recombinantly produced bacteriophageproteins. This coupling can be effected directed, e.g. on the N- orC-terminus or be undirected. The directed coupling is effected via asuitable, reactive amino acid, such as cysteine, which is of course notfrequently surface-exposed in phage proteins and has been introducedspecifically at a suitable position. Since phage tail proteins aresynthesised in the cytoplasma, disulfide bridges do not need to be takeninto account. Preferably, coupling can take place also via other aminoacids, directly or as also with cysteine indirectly via a “spacer” or“cross linker” (monofunctional or bifunctional).

In the case of cysteine coupling, all bifunctional crosslinkers with NH-and SH-reactive groups are possible, with and without intermediatespacers, e.g. 11-maleimidoundecanoic acid sulfo-NHS orsuccinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxy-[6-amido]caproate.If no spacers are present, 8-12 C-atom-spacers with a terminal NH groupcan be inserted. Preferably the cysteine coupling is effected via aspecific biotinylation of cysteine by for example EZ-link-PEO-maleimideactivated biotin (Pierce).

Bivalent ions, such as e.g. Ca²⁺ or Mg²⁺ are important for bindingendotoxins to phage proteins, such as p12. By adding suitable chelatingagents, such as e.g. EDTA or EGTA, this binding can however be broken.For the binding, Ca²⁺ concentrations are preferred in the range ofapproximately 0.1 μM to approximately 100 mM, particularly preferred inthe range of approximately 0.1 μM to approximately 10 mM, and especiallypreferred in the range of approximately 0.1 μM to approximately 1 mM andfurthermore particularly preferred in the range of approximately 10 μMto 1 mM. If the concentration of bivalent ions is lowered by adding 1 mMEDTA under 100 nM, then the binding of endotoxin to p12 is broken. Mg²⁺concentrations above 10 mM make the binding of endotoxin to p12 worse,which becomes noticeable in an increase in the dissociation constant.Without addition of Mg²⁺, a K_(d) value of 50 nM is produced and, in abuffer with 10 mM Mg²⁺, a K_(d) value of 1 μM was measured. Zincrevealed an even higher inhibiting effect. 1 mM Zn increases the K_(d)value to 10 μM. An adjustment of the concentration of bivalent or otherions (e.g.: Cu²⁺, Al³⁺, Zn²⁺, Fe²⁺, Ca²⁺, Ba²⁺, Mg²⁺, Cd²⁺) to a rangewhich is optimal for the binding, can be effected by substances such asHEDTA, NTA or general chelating agents/buffers (ADA:N-[2-acetamido]-2-iminodiacetic acid; 5-AMP: adenosine-5′-monophosphate;ADP: adenosine-5′-diphosphate; ATP: adenosine-5′-triphosphate; Bapta:1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′,-tetraacetic acid; citrate:citric acid; EDTA: ethylene diamine tetraacetic acid; EGTA:ethyleneglycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid;HEDTA: N-hydroxyethylethylenediaminetriacetic acid; NTA:nitrilotriacetic acid; SO₄ sulfate), which can be used as buffers forbivalent ions.

The methods according to the invention can therefore comprise furtherwashing steps. According to whether a direct or indirect detection orthe depletion requires separation of sample and bacteriophage tailprotein, washing steps can be incorporated. Since Ca²⁺ or other metalions (e.g. Mg²⁺) are essential for the binding, the binding of endotoxinto e.g. p12 can be broken by suitable washing steps. According to theaim of whether endotoxin is intended to remain bonded on thebacteriophage tail protein, e.g. p12, washing takes place with EDTA-freebuffer, if the binding is intended to be broken, with EDTA-containingbuffer, the EDTA concentrations being in the range of at least 0.05 mMto more than 10 mM, preferably in the range of 2 mM to 5 mM.

The separation is effected after incubation of the sample with thecarrier material, which is coupled correspondingly with bacteriophagetail proteins, for approximately 5-60 min or approximately 30-180 minor, if required, also overnight. For this purpose, the sample is elutede.g. from the chromatography column, or filtered or the correspondingparticles are centrifuged off or sedimented off or are separatedmagnetically by applying a magnetic field. The separation in the batchmethod described here, i.e. with pre-incubation of sample and carriermaterials, which are coupled with the corresponding bacteriophage tailproteins, can be sensible in particular with very low endotoxinconcentrations.

The depletion of endotoxins via chromatography columns can however alsobe effected in the pure throughflow method. The sample can be applied tothe column for this purpose, which column contains a carrier materialwith bacteriophage tail proteins coupled thereto. The flow rate isdependent upon the volume and geometry of the column. The flow rate isfurthermore dependent upon the volume and endotoxin content of thesample in order to achieve, by means of as long a contact time aspossible between column and endotoxin, even in the case of low endotoxinconcentrations, an efficient depletion. The contact time is thereby thetime which the sample requires from application on the column untilflowing out.

The separation step can be used for example in the depletion method toregenerate the bacteriophage tail proteins which are coupled to thepermanent carrier. As a result, the permanent carrier, e.g. a matrix,can be recycled in a chromatography column. Regeneration is effected byremoving the bonded endotoxin by means of a suitable regeneration buffercontaining EDTA or a corresponding chelating agent. In the case of EDTA,a concentration of greater than 2 mM EDTA is preferred, in particulargreater than 10 mM EDTA.

Since ionic interactions can fundamentally always be affected by changesin the ion strength, increases or reductions of other salts in thesolution, such as e.g. NaCl or KCl, can also affect the binding ofendotoxin to the bacteriophage tail proteins.

In order to make the binding visible directly or indirectly in thedetection method, the protein can also be altered molecular-biologicallyor biochemically in order to enable measurement or to improve it. Inorder to make binding of endotoxin e.g. to p12 directly visible, amolecular-biological exchange of tyrosine radicals for tryptophan can beimplemented. It can thereby be necessary for a reduction in the signalbackground to exchange the originally contained tryptophans fortyrosines. In order to be able to make measurements also inprotein-containing solutions, p12 can be modified chemically in additionafter tryptophan introduction. Tryptophan radicals are thereby alteredby Koshland reagent (2-hydroxy-5-nitrobenzylbromide) with respect totheir spectroscopic properties. In the case of displacement experiments,marked, e.g. fluorescence-marked endotoxin (e.g. Sigma) can be displacedby endotoxin, e.g. by p12, which is located in the sample and theconcentration of free fluorescent endotoxin can be determined.

With the method according to the invention, endotoxin can be detected inand removed from all aqueous solutions. These solutions can contain:proteins, plasmid-DNA, genomic DNA, RNA, protein-nucleic acid complexes,such as e.g. phages or viruses, saccharides, vaccines, drugs, dialysisbuffers (medicine), salts or other substances contaminated by endotoxinbinding.

A further aspect of the invention is bacteriophage proteins, to whichthe so-called tags, e.g. the Strep- or His-tag, are coupled preferablyto the N- or C-terminus of the protein, particularly preferred to theC-terminus. The coupling or cross-linking of the tags with thebacteriophage proteins via DNA recombination technology is preferred.Production of the nucleic acid, comprising the sequence of thebacteriophage protein and of the tag and the production of theexpression product are the state of the art and do not require to beexplained here separately. A further aspect of the invention is thenucleic acid sequence which encodes a bacteriophage protein togetherwith the Strep- or His-tag. The p12 protein of the phage T4 is aparticularly preferred bacteriophage protein which is modified with theStrep- or His-tag but all other bacteriophage proteins, which areinvolved in detection and binding of bacteria or are responsible forthis, are likewise preferred.

A further aspect of the invention is bacteriophage proteins with a tagwhich has a surface-exposed cysteine for specific directedbiotinylation, e.g. the tags according to SEQ ID NO: 5, 6 and 7. Anexample of a p12 with a tag is the amino acid sequence cited in SEQ IDNO: 8. A p12 with a tag is preferred, in particular with a tag with asurface-exposed cysteine, in particular a p12 with the tag according toSEQ ID NO: 6 and 7. This directed biotinylation can be imparted inaddition by a suitable spacer or linker. Furthermore, the presentinvention relates to the amino acids with a sequence according to SEQ IDNO: 5, 6 and 7. Furthermore, the present invention relates to thenucleic acids which encode the amino acid sequence according to SEQ IDNO: 5, 6 and 7.

The methods according to the invention, relative to detection andpurification methods for and of endotoxin, offer advantages in theperformance of corresponding applications. Furthermore, the productionof antibodies against LPS core oligosaccharides is very difficult, whichrenders corresponding methods based on antibodies very expensive.

The following examples explain the invention and should not beunderstood as restrictive. If not otherwise indicated,molecular-biological standard methods were used, such as e.g. describedby Sambrook et al., 1989, Molecular cloning: A Laboratory Manual 2^(nd)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

1. Glass Vessels, Plastic Vessels and Buffers

For the endotoxin removal, all the glass vessels were depyrogenated byheating at 200° C. (4 h) and exclusively pyrogene-free plastic materials(e.g. pipette tips, microtitre plates) were used. Other non-heatresistant appliances or vessels were treated either with 3% hydrogenperoxide or washed with 1% sodium deoxycholate. Subsequently, they wererinsed with endotoxin-free water. The buffers were produced fromextensively endotoxin-free buffer substances (Sigma) and mixed withendotoxin-free water. Salts, such as e.g. NaCl, which can be heated to200° C., were heated up (200° C., 4 h). Buffers used for chromatographicpurifications were degassed and filtered.

2. Endotoxin Detection by Means of LAL Test

Endotoxin control tests were implemented with a chromogenic LAL test(Limulus-Amoebocyte-Lysate test, Charles-River Endosafe, Charleston,USA) corresponding to the instructions of the producer. In order todetermine the concentrations, endotoxin standards (Charles-RiverEndosafe, Charleston, USA) in the range of 0.005-50 or 0.02-50 EU/mlwere used. The absorption measurement at 405 nm took place in atemperature-controlled microtitre plate reader (Genios, Tecan GmbH).

3. Western-Blot for p12 Detection

The detection of p12 in the residue of samples treated with beads or inthe fractions of the affinity chromatography was effected by WesternBlots. In part, the proteins were concentrated in advance by NaDOC/TCAprecipitation (sodium deoxycholate/tetrachloroacetate). The samples wereelectrophoretically separated for this purpose on 12% SDS gels andtransferred onto PVDF membranes (Immobilon, Millipore). The membraneswere washed with PBS for 30 min, blocked with 5% milk powder (1 h) andsubsequently incubated with polyclonal anti-p12 antibody (1 h, dilution:1:1000). After incubation with a secondary antibody (goat-anti-rabbitIgG), conjugated with alkaline phosphatase, the development of thesamples was effected with BCIP/NBT(5-bromo-4-chloroindolylphosphate/nitroblue tetrazolium salt).

4. Endotoxin Purification

The purification of endotoxin was implemented according to thespecification of Galanos, C., Lüderitz, O. & Westphal, O. 1969, Europ.J. Biochem. 9, 245-249.

EXAMPLE 5 Specific Coupling of p12 to Immobilised Iodoacetyl Radicals

In order to achieve a directed binding of p12 to the surface, the aminoacid serin at position 3 of the Strep-tag according to SEQ ID NO:5 wasreplaced by cysteine as in example 12 and the protein was immobilisedvia iodoacetyl radicals which bind preferably free sulfydryl radicals.The resulting p12 was called p12S3C.

A 1 ml Sulfolink Coupling Gel (Pierce) was poured out, washed with 6 ml1% sodium deoxycholate and equilibrated with 6 ml coupling buffer (50 mMtris, 150 mM NaCl, 5 mM EDTA, pH 8.5). Subsequently, 1 ml p12S3C(=N-strepS3Cp12) was injected (1-1.5 mg/ml in coupling buffer), thecolumn was agitated gently for 15 min, incubated for a further 30 minwithout agitation at room temperature, and 1 ml p12S3C was injectedagain and the incubation steps were repeated. This coupling of p12S3Cwas repeated in total 4 times, and subsequently the column was washedwith 6 ml coupling buffer. The throughflows were collected and therespective p12S3C concentration was determined by absorption measurementat 280 nm. 2.2-2.8 mg p12S3C per ml gel were bonded. Subsequently,surplus iodoacetyl radicals were blocked by incubation (45 min) with 1ml cysteine (50 mM in 50 mM tris, 5 mM EDTA, pH 8.5). After washing thecolumn with 16 ml 1M NaCl and 16 ml 20 mM hepes, 150 mM NaCl pH 7.5, thecolumn was ready for use.

The capacity of this gel to remove endotoxin from protein solutions wastested with BSA (2-4 mg/ml), carbonic anhydrase (1-2 mg/ml) and lysozyme(3-4 mg/ml). BSA and lysozyme solutions were spiked with endotoxin fromE. coli O55:B5 (Charles-River Endosafe, Charleston, USA) or E. coli HMS174 (100-1000 EU/ml), whilst the carbonic anhydrase was not mixed withadditional endotoxin. Respectively 0.5 ml protein solution wasintroduced to the column, incubated for 1 hour at room temperature andsubsequently the column was washed with buffer. The proteins werecollected in fractions and the endotoxin content, prior to and after thecolumn, was determined by means of a chromogenic LAL test (Charles-RiverEndosafe, Charleston, USA). In addition, the protein retrieval wasdetermined by absorption measurements at 280 nm. The endotoxins wereable to be removed almost completely (93-99%) from all 3 proteinsolutions, as shown in FIG. 2A. In addition, the proteins were able tobe eluted extensively from the column (80-99%, FIG. 2B). The column wasfinally regenerated with 5 mM EDTA, 20 mM hepes, 150 mM NaCl, pH 7.5. Inorder to exclude impurities of the protein fractions after running overthe column due to separating p12, the fractions were tested for p12 bymeans of the Western Blot technique. No p12 was able to be detected inthe fractions.

EXAMPLE 6 Non-Specific Coupling of p12 to NHS-Activated Carrier Material

N-hydroxysuccinimide (NHS) is displaced from compounds by primary aminoradicals and therefore is used to couple proteins to surfaces.NHS-activated sepharose columns (HiTrap NHS-activated HP, 1 ml,Amersham-Pharmacia-Biotech) were washed firstly with 6 ml ice cold 1 mMhydrochloric acid. Subsequently, 10-15 ml p12S3C (1.0-3.5 mg/ml) in 0.2M NaHCO₃, 0.5 M NaCl, pH 8.3 were pumped in a circle over the column atroom temperature (flow rate 0.8 ml/min). After 60 min, the throughflowwas collected in fractions and the column was washed with 6 ml buffer.From these fractions, the NHS was separated by desalting the solutionvia HiTrap-desalting column (5 ml, Amersham-Pharmacia-Biotech) andsubsequently the p12 quantity was determined by absorption measurementat 280 nm. 20-25 mg p12S3C were bonded to the column. The column wasrinsed after the coupling corresponding to the instructions of theproducer repeatedly with respectively 6 ml blocking buffer (0.5 Methanolamine, 0.5 M NaCl, pH 8.3) and washing buffer (0.1 M acetate, 0.5M NaCl, pH 4.0). Subsequently, the column was equilibrated with 6 mlusable buffer (20 mM hepes, 150 mM NaCl, pH 7.5 or 20 mM tris, 150 mMNaCl, pH 8.5).

The endotoxin removal via this column was tested with lysozyme solutions(3-4 mg/ml in 20 mM hepes, 150 mM NaCl, pH 7.5 or 20 mM tris, 150 mMNaCl, pH 8.5). The lysozyme solutions were spiked with endotoxin from E.coli HMS 174 (˜500 EU/ml). 0.5 ml protein solution were introduced ontothe column, incubated for 1 hour at room temperature and subsequentlythe column was washed with buffer. The lysozyme was collected infractions and the endotoxin content was determined prior to and afterthe column by means of a chromogenic LAL test (Charles-River Endosafe,Charleston, USA). In addition, the protein retrieval was determined byabsorption measurements at 280 nm. The endotoxins were removed up to85-90% from the solution, as shown in FIG. 3A, and 85-90% of thelysozyme were able to be eluted again from the column by means ofwashing with usable buffer (FIG. 3B). The column was subsequently washedwith 6 ml 5 mM EDTA, 20 mM hepes, 150 mM NaCl, pH 7.5 and 6 ml 1 M NaCl.In order to exclude impurities of the protein fractions after runningover the column due to separating p12, the fractions were tested bymeans of the Western Blot technique for p12. No p12 was able be detectedin the fractions.

EXAMPLE 7 Directed Coupling of p12 to NHS-Activated Carrier MaterialColumn Via Diaminoethane and N-succinimidyl-iodoacetate (SIA) as Spacer

In order to achieve a directed binding to the chromatography carriermaterial, a bifunctional linker was bonded to NHS-activated surface,which linker made a coupling of p12S3C possible via its free cysteineand iodoacetyl radicals of the bifunctional linker.

NHS-activated sepharose columns (HiTrap NHS-activated HP, 1 mlAmersham-Pharmacia-Biotech) were washed firstly with 6 ml ice cold 1 mMhydrochloric acid, thereafter 1 ml ethylene diamine (10 mg/ml in 0.2 MNaHCO₃, 0.5 M NaCl, pH 8.3) was injected and the column was incubatedfor 30 min at room temperature. After blocking surplus NHS groups withethanolamine (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and washing (0.1 Macetate, 0.5 M NaCl, pH 4.0) of the column, the column was equilibratedwith 6 ml borate buffer (50 mM sodium borate, 150 mM NaCl, 5 mM EDTA, pH8.3). Subsequently, 10 ml N-succinimidyl-iodoacetate (SIA, Pierce, 200μl SIA parent solution in 10 ml borate buffer; SIA parent solution: 1.4mg SIA in 1 ml DMSO) was rinsed in a circle over the column for 30 min.The column was thereafter washed with 6 ml borate buffer and p12S3C (1mg/ml, 50 ml in borate buffer) was rinsed over the column for 1 hour.Excess iodoacetyl radicals were neutralised with 1 ml cysteine solution(5 mM cysteine in borate buffer, incubation at room temperature for 15min), before the column with the usable buffers (20 mM hepes, 150 mMNaCl, pH 7.5 or 50 mM tris, 150 mM NaCl, ph 8.5) were equilibrated. Thecoupling reactions with SIA were implemented in the dark.

The endotoxin removal over this column was tested with lysozymesolutions (3-4 mg/ml in 20 mM hepes, 150 mM NaCl, pH 7.5 or 20 mM tris,150 mM NaCl, ph 8.5). The lysozyme solutions were spiked with endotoxinfrom E. coli HMS 174 (˜500 EU/ml). 0.5 ml protein solution wasintroduced onto the column, was incubated for 1 hour at room temperatureand subsequently the column was washed with buffer. The lysozyme wascollected in fractions and the endotoxin content was determined prior toand after the column by means of a chromogenic LAL test (Charles-RiverEndosafe, Charleston, USA). In addition, the protein retrieval wasdetermined by absorption measurements at 280 nm. The endotoxins wereremoved up to 90% from the solution, as shown in FIG. 3A, and 75-85% ofthe lysozyme were able to be eluted again from the column by washingwith usable buffer (FIG. 3B). The column was subsequently washed with 6ml 5 mM EDTA, 20 mM hepes, 150 mM NaCl, pH 7.5 and 6 ml 1 M NaCl. Inorder to exclude impurities of the protein fractions after running overthe column due to separating p12, the fractions were tested for p12 bymeans of the Western Blot technique. No p12 was able to be detected inthe fractions.

EXAMPLE 8 Removal of Endotoxin From a BSA Solution in the ThroughflowMethod

HiTrap-NHS activated sepharose (Amersham Biosciences, Uppsala Sweden)was coupled, according to the specification of the producer,non-specifically via primary amino groups with p12. 8 mg p12/ml gelmaterial were thereby immobilised covalently. The thus obtained 1 mlchromatography column was equilibrated with a flow rate of 1 ml/min with10 ml buffer A (20 mM hepes, pH 7.5, 150 mM NaCl, 0.1 mM CaCl₂). Next, 4ml of a BSA solution (11.5 mg BSA (Carl Roth GmbH, Germany)/ml buffer A)were applied (injection: I) and the throughflow (E) was collected in 2.5ml fractions. The column was washed subsequently with 15 ml buffer A andthe endotoxin bonded to the column was eluted with 7 ml buffer B (20 mMhepes, pH 7.5, 150 mM NaCl, 2 mM EDTA). During washing and elution,respectively 2 ml fractions were collected. After each experiment, thecolumn was regenerated with 20 ml buffer C (20 mM hepes, pH 7.5, 150 mMNaCl, 2 mM EDTA, 0.1% sodium deoxycholate). The endotoxin concentrationwas determined by a chromogenic Limulus Amoebocyte Lysate (LAL)(Charles-River Endosafe, Charleston, USA) according to the specificationof the producer. Determination of the protein concentration was effectedby measurement of the UV absorption. The endotoxin removal efficiencywas between 95-99% and the protein loss was approximately 6-10%.

EXAMPLE 9 Removal of Small Endotoxin Quantities From Buffer by Means ofNon-Specifically Coupled p12

20 ml NHS-activated sepharose 4 FastFlow (Amersham Biosciences) werewashed firstly with ice cold hydrochloric acid and subsequentlyincubated with 292 mg p12 (7 mg/ml in 25 mM citrate pH 7.0) for 4 hoursat room temperature with agitation. Subsequently, the sepharose waswashed with 7×80 ml 5 mM citrate pH 2.0 and respectively 1 ml of thewashing fractions was dialysed against 5 mM citrate pH 2.0. Thesedialysates were used in order to quantify the excess p12 in the washingfractions by means of absorption measurement at 280 nm. A charge densityof 8.7 mg p12 per 1 ml sepharose was determined. Non-reacted NHSradicals were neutralised by 12 h incubation of the sepharose with 1Mtris pH 8.0. Columns with 2 ml volume were filled with this columnmaterial and this was stored until use at 4° C. in 20% ethanol.

In 3 parallel tests, respectively 4 ml endotoxin solution (S) wereapplied onto a column (see FIG. 9). The endotoxin solution comprisedendotoxin from E. coli O55:B5 (Charles-River Endosafe, Charleston, USA)in equilibration buffer (20 mM hepes, 150 mM NaCl, 0.1 mM CaCl₂, pH7.5). The endotoxin concentration of this solution was 4.6 EU/ml.

The column was rinsed firstly with 12 ml regeneration buffer (20 mMhepes, 150 mM NaCl, 2 mM EDTA, pH 7.5) and subsequently with 12 mlequilibration buffer. Subsequently, equilibration buffer was introducedonce again to the column and 1 ml was fractionated.

The endotoxin solution was applied onto the columns (I) and fractions of5 ml and 2 ml were collected. Subsequently, the column was regeneratedwith 4 ml regeneration buffer (B). In the throughflow fractions, noendotoxin could be detected, i.e. the endotoxin impurities were able tobe removed completely in all three experiments.

EXAMPLE 10 Non-Specific Coupling of Biotinylated p12 to MagneticStreptavidin Beads

p12 (3 mg/ml in PBS, 0.05% Tween20) was incubated withsulfo-NHS-LC-LC-biotin (Pierce), in the ratio 1:10 to 1:20 for 1 hour atRT and subsequently was dialysed against buffer (e.g. PBS or 20 mMhepes, 150 mM NaCl, 5 mM EDTA, pH 7.5). NHS-activated biotin bindsthereby to primary amino radicals of p12. Subsequently 50 μlbiotinylated p12 (1 mg/ml) were added to 1 ml streptavidin beads(MagPrep streptavidin beads, Merck), were agitated at room temperaturefor 2 h and subsequently excess p12 was removed by washing four timeswith 1.5 ml 20 mM tris, 10 mM EDTA, pH 7.5.

The endotoxin removal was tested with buffer (20 mM hepes, 150 mM NaCl,pH 7.5) and protein solutions (0.1 mg/ml BSA, 0.1 mg/ml lysozyme, 0.1mg/ml carbonic anhydrase in 20 mM hepes, 150 mM NaCl, pH 7.5). Thebuffer and the BSA and lysozyme solution was spiked with 5 EU/ml(endotoxin from E. coli O55:B5, Charles-River Endosafe, Charleston,USA). The carbonic anhydrase solution contained approximately 1 EU/ml.25 μl magnetic beads with immobilised p12 were added to 200 μl buffer orprotein solution, mixed by pipetting up and down and were incubated for30 min at room temperature. The beads were removed from the solution bymeans of a magnet, the residue was pipetted off. The endotoxin contentof untreated samples and samples incubated with beads was subsequentlydetermined with the LAL test and the protein retrieval was determined byabsorption measurement at 280 nm. The endotoxin could be practicallycompletely removed from the buffer (99.9% endotoxin removal, FIG. 4A)and the endotoxin was depleted also from the protein solution by 70-92%(FIG. 4B). The protein retrieval was between 57% and 99% (BSA: 87%,carbonic anhydrase: 99%, lysozyme: 57%; FIG. 4B).

EXAMPLE 11 Non-Specific Coupling of Biotinylated p12 to ImmobilisedStreptavidin

p12 (3 mg/ml in PBS, 0.05% Tween20) was incubated withsulfo-NHS-LC-LC-biotin (Pierce), in the ratio 1:10 to 1:20 for one hourat RT and subsequently dialysed against buffer (e.g. PBS or 20 mM hepes,150 mM NaCl, 5 mM EDTA, pH 7.5). NHS-activated biotin thereby binds toprimary amino radicals of p12. The biotinylated p12 is subsequentlyincubated for 1 h at room temperature with chromatography material ladenwith streptavidin (Immunopure immobilised streptavidin: 6% cross-linkedagarose beads) and excess p12 is removed by washing with PBS.

The endotoxin removal was tested with buffer (20 mM tris, 150 mM NaCl,pH 8.0) and BSA (0.5 mg/ml in 20 mM tris, 150 mM NaCl, pH 8.0).Respectively 1 ml buffer or BSA solution was spiked with 10 EU/ml, 50 μlp12 agarose was added, agitation took place for 1 hour at roomtemperature. The p12 agarose was centrifuged off subsequently and theendotoxin- and protein concentration in the residue was measured. 99%endotoxin could be removed from the buffer and 86% from the BSA solution(FIG. 5). BSA was retrieved up to 90%.

EXAMPLE 12 Tests via p12 Endotoxin Binding by Means of Surface PlasmonResonance Measurements

Binding of p12 to endotoxin or to bacteria via the liposaccharides inthe outer cell membrane was tested by means of surface plasmon resonancemeasurements (Biacore J). In order to determine the dissociationconstant (K_(d)), endotoxin from E. coli O55:B5 (Sigma) was immobilisedon a hydrophobic HPA chip corresponding to the instructions of theproducer and p12 was injected in various concentrations (FIG. 6A).Binding is measured in relative “response units” (RU), the equilibriumvalues are plotted against the associated p12 concentrations (FIG. 6B).By adapting the Langmuir adsorption isotherms(RU=(RU_(max)*[p12])/([p12]+K_(d))) to these data, the K_(d) value wasdetermined (Table 1). Endotoxin-free buffers were used for themeasurements. K_(d) values in the range of 10⁻⁷ to 10⁻⁹ M weredetermined for pH values between 6 and 10 (Table 1). The binding wasbroken again by injection of 1 mM or 5 mM EDTA and the chip wasregenerated. TABLE 1 Dissociation constants of endotoxin on p12dependent upon the pH value of the solution pH Kd 6.00 3.09E−07 7.506.85E−08 8.00 5.86E−08 8.50 7.86E−08 9.00 3.29E−08 10.00 1.55E−07

In order to test the binding of bacteria to p12, biotinylated p12 wasimmobilised on streptavidin chips and various E. coli strains wereinjected. The bacteria were absorbed in PBS for the measurements. E.coli strains were used which have lipopolysaccharides with differentpolysaccharide components. The polysaccharide part comprises a “core”region which is cross-linked to the lipid A and to the so-called Oantigen. The O antigen varies very greatly between different types ofbacteria and also strains of bacteria, whilst the “core” region ishighly preserved. Strains, which have the “core” region and O antigen(e.g. E. coli), and strains which have a complete “core” region (E. coliD21), were bonded by p12, whilst strains with a greatly shortened “core”region (e.g. E. coli D21f2) were no longer detected by p12 (FIG. 6C).The binding was able to be broken again by EDTA (5 mM) and the chip wasable to be regenerated.

EXAMPLE 13 Recombinant p12 Constructs

1. Construction of p12 with N-terminal Strep-tag (N-strep-p12): by meansof PCR, the nucleotide sequence for the Strep-tag (U.S. Pat. No.5,506,121) was introduced to the 5′ end of the T4p12 gene. A primer wasconstructed for this purpose for the 5′ end of the p12 gene (5′-GAA GGAACT AGT CAT ATG GCTAGC TGG AGC CAC CCG CAG TTC GAA AAA GGC GCC AGT AATAAT ACA TAT CAA CAC GTT-3′ (SEQ ID NO:1), which comprises the nucleotidesequence of the Strep-tag at its 5′ end (italicised in the sequence) andhas a restriction interface (NdeI, underlined in the sequence) such thatthe gene in the right-hand reading grid can be inserted into theexpression plasmid. For the 3′ end of the p12 gene, a primer wasconstructed which introduces, behind the p12 gene, a BamH I restrictioninterface (italicised in the sequence) (5′-ACG CGC AAA GCT TGT CGA CGGATC CTA TCA TTC TTT TAC CTT AAT TAT GTA GTT-3′), (SEQ ID NO:2). The PCRwas implemented with 40 cycles (1 min 95° C., 1 min 45° C. and 1 min 72°C.). The PCR batch was cut with the restriction endonucleases NdeI andBamHI and the desired fragment was inserted after size fractionation viaan agarose gel and elution from the gel into the NdeI and BamHI site ofthe expression plasmid pET21a. The sequence of the N-strep-p12 gene waschecked for its correctness via DNA sequencing. The further steps forthe plasmid pNS-T4p12p57 were implemented as described by Burda, M. R. &Miller, S. (Eur J Biochem. 1999 265 (2), 771-778) for T4p12p57. Theplasmid pNS-T4p12p57 was then transformed into the expression strainBL21(DE3).

2. Insertion of an N-terminal cysteine radical in N-strep-p12(N-strep-S3C-p12 and N-strep-S14C-p12): the insertion of an N-terminalcysteine radical was implemented as described under 1, two new primersfor the 5′ end being constructed for this purpose. There was used forthe N-strep-S3C-p12, the primer 5′-GAA GGA ACT AGT CAT ATG GCT TGT TGGAGC CAC CCG CAG TTC GAA AAA GGC GCC AGT AAT AAT ACA TAT CAA CAC GTT-3′(SEQ ID NO:3), there was used for the N-strep-S14C-p12, the primer5′-GAA GGA ACT AGT CAT ATG GCTAGC TGG AGC CAC CCG CAG TTC GAA AAA GGCGCC TGT AAT AAT ACA TAT CAA CAC GTT-3′ (SEQ ID NO:4).

3. Purification of N-strep-p12 protein: the E. coli strain BL21(DE3)with the plasmid pNS-T4p12p57 was drawn in 2 1 shaker cultures (LBmedium with ampicillin 100 μg/ml) up to a OD600 of 0.5-0.7 at 37° C. andthe expression of the N-strep-p12-protein was induced by addition of 1mM IPTG (isopropyl-β-thio-galactopyranoside). After incubation at 37° C.for 4 h, the cells were collected. Collected cells from 10 1 culturewere taken up in 50 ml sodium phosphate, 20 mM pH 7.2, 2 mM MgSO4, 0.1 MNaCl, broken up by French press treatment (20,000 psi) three times andsubsequently centrifuged off for 30 min at 15,000 rpm (SS34). Afterwashing twice in the same buffer, the N-strep-p12 protein was extractedfrom the pellet, the pellet was extracted three times by agitation for30 min in 40 mM trisHCl pH 8.0, 10 mM EDTA, the batch was centrifugedfor 30 min at 15,000 rpm (SS34) and the dissolved NS-p12 was stored inthe residue at 4° C. The extraction was repeated twice and the combinedresidues were applied (IBA GmbH Göttingen) onto a StrepTactin affinitycolumn (15 ml), equilibrated with buffer “W” (100 mM trisHCl pH 8, 1 mMEDTA, 150 mM NaCl). After washing with 5 column volumes of buffer “W”,elution took place with three volumes of buffer “W” with 2.5 mMdethiobiotin in buffer “W”. After multiple dialysis against buffer “W”and concentration, the concentration and purity of N-strep-T4p12 wasdetermined via SDS-PAGE and UV spectroscopy (Burda et al. 1999). From 10litres culture, approximately 100 mg N-strep-T4p12 were thus purified.Name Sequence of the tag Nstrep-p12 MASWSHPQFEKGAS SEQ ID NO: 5Nstrep-p12-S3C MACWSHPQFEKGAS SEQ ID NO: 6 Nstrep-p12-S14CMASWSHPQFEKGAC SEQ ID NO: 7

1. A method for detecting endotoxin, comprising the steps: a) incubatinga sample with a bacteriophage tail protein, and b) detecting endotoxinbonded to bacteriophage tail proteins.
 2. The method according to claim1, further comprising after step a) and prior to step b) the additionalstep of: a′) separating the bacteriophage tail protein-endotoxincomplexes from the sample.
 3. The method according to claim 1, whereindetection comprises spectroscopic methods.
 4. A method for removingendotoxin from a sample, comprising the steps: a) incubating a samplewith or bringing a sample in contact with bacteriophage tail proteinswhich are immobilised on a permanent carrier, non specifically ordirected, b) separating the bacteriophage tail protein-endotoxin complexfrom the sample.
 5. The method according to claim 4, wherein steps a)and b) are implemented in a chromatography column throughflow method. 6.The method according to claim 4, wherein the permanent carrier comprisesfiltration media, glass particles, magnetic particles, centrifugationmaterials, sedimentation materials or filling materials forchromatography columns.
 7. The method according to claim 4, thebacteriophage tail proteins being immobilised on the permanent carriervia coupling groups.
 8. The method according to claim 7, the couplinggroup being a lectin, receptor or anticalin.
 9. The method according toclaim 7, wherein the coupling group comprises streptavidin or avidin andthe bacteriophage tail proteins is coupled with biotin or a Strep-tag.10. The method according to claim 4, the bacteriophage tail proteinsbeing immobilised on the permanent carrier covalently via chemicalbonds.
 11. The method according to claim 1, wherein the bacteriophagetail protein comprises a Strep-tag or a His-tag.
 12. The methodaccording to claim 11, wherein the tag comprises an amino acid sequenceaccording to SEQ ID NO. 5, 6 or
 7. 13. The method according claim 11,wherein the p12 protein of the phage T4 is used as bacteriophage tailprotein.
 14. The method according to claim 1, wherein the Ca²⁺concentration of the incubation comprises 0.1 μM to 10 mM and the Mg²⁺concentration comprises 0.1 μM to 10 mM.
 15. The method according to oneof the claim 1, marked endotoxin being displaced from the bond with abacteriophage tail protein and the marked endotoxin being subsequentlydetected.
 16. The method according to claim 1, wherein the bacteriophagetail protein comprises a Strep-tag or a His-tag.
 17. The methodaccording to claim 16, wherein the tag comprises an amino acid sequenceaccording to SEQ ID NO. 5, 6 or
 7. 18. The method according claim 16,wherein the p12 protein of the phage T4 being used as bacteriophage tailprotein.